Cytokine-facilitated transduction leads to low-level engraftment in nonablated hosts

Using a murine bone marrow transplantation model, we evaluated the long-term engraftment of retrovirally transduced bone marrow cells in nonmyeloablated hosts. Male bone marrow was stimulated in a cocktail of interleukin-3 (IL-3), IL-6, IL-11, and stem cell factor (SCF) for 48 hours, then cocultured on the retroviral producer line MDR18.1 for an additional 24 hours. Functional transduction of hematopoietic progenitors was detected in vitro by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of multiple drug resistance 1 (MDR1) mRNA from high proliferative potential-colony forming cell (HPP-CFC) colonies. After retroviral transduction, male bone marrow cells were injected into nonablated female mice. Transplant recipients received three TAXOL (Bristol-Myers, Princeton, NJ) injections (10 mg/kg) over a 14-month period. Transplant recipient tissues were analyzed by Southern blot and fluorescence in situ hybridization for Y-chromosome-specific sequences and showed donor cell engraftment of approximately 9%. However, polymerase chain reaction amplification of DNAs from bone marrow, spleen, and peripheral blood showed no evidence of the transduced MDR1 gene. RT-PCR analysis of total bone marrow RNA showed that transcripts from the MDR1 gene were present in a fraction of the engrafted donor cells. These data show functional transfer of the MDR1 gene into nonmyeloablated murine hosts. However, the high rates of in vitro transduction into HPP-CFC, coupled with the low in vivo engraftment rate of donor cells containing the MDR1 gene, suggest that the majority of stem cells that incorporated the retroviral construct did not stably engraft in the host. Based on additional studies that indicate that ex vivo culture of bone marrow induces an engraftment defect concomitantly with progression of cells through S phase, we propose that the cell cycle transit required for proviral integration reduces or impairs the ability of transduced cells to stably engraft

A Synthetic Interaction Screen Identifies Factors Selectively Required for Proliferation and TERT Transcription in p53-Deficient Human Cancer Cells

Numerous genetic and epigenetic alterations render cancer cells selectively dependent on specific genes and regulatory pathways, and represent potential vulnerabilities that can be therapeutically exploited. Here we describe an RNA interference (RNAi)-based synthetic interaction screen to identify genes preferentially required for proliferation of p53-deficient (p53-) human cancer cells. We find that compared to p53-competent (p53+) human cancer cell lines, diverse p53- human cancer cell lines are preferentially sensitive to loss of the transcription factor ETV1 and the DNA damage kinase ATR. In p53- cells, RNAi-mediated knockdown of ETV1 or ATR results in decreased expression of the telomerase catalytic subunit TERT leading to growth arrest, which can be reversed by ectopic TERT expression. Chromatin immunoprecipitation analysis reveals that ETV1 binds to a region downstream of the TERT transcriptional start-site in p53- but not p53+ cells. We find that the role of ATR is to phosphorylate and thereby stabilize ETV1. Our collective results identify a regulatory pathway involving ETV1, ATR, and TERT that is preferentially important for proliferation of diverse p53- cancer cells

Comparative whole genome sequencing reveals phenotypic tRNA gene duplication in spontaneous Schizosaccharomyces pombe La mutants

We used a genetic screen based on tRNA-mediated suppression (TMS) in a Schizosaccharomyces pombe La protein (Sla1p) mutant. Suppressor pre-tRNASerUCA-C47:6U with a debilitating substitution in its variable arm fails to produce tRNA in a sla1-rrm mutant deficient for RNA chaperone-like activity. The parent strain and spontaneous mutant were analyzed using Solexa sequencing. One synonymous single-nucleotide polymorphism (SNP), unrelated to the phenotype, was identified. Further sequence analyses found a duplication of the tRNASerUCA-C47:6U gene, which was shown to cause the phenotype. Ninety percent of 28 isolated mutants contain duplicated tRNASerUCA-C47:6U genes. The tRNA gene duplication led to a disproportionately large increase in tRNASerUCA-C47:6U levels in sla1-rrm but not sla1-null cells, consistent with non-specific low-affinity interactions contributing to the RNA chaperone-like activity of La, similar to other RNA chaperones. Our analysis also identified 24 SNPs between ours and S. pombe 972h- strain yFS101 that was recently sequenced using Solexa. By including mitochondrial (mt) DNA in our analysis, overall coverage increased from 52% to 96%. mtDNA from our strain and yFS101 shared 14 mtSNPs relative to a ‘reference’ mtDNA, providing the first identification of these S. pombe mtDNA discrepancies. Thus, strain-specific and spontaneous phenotypic mutations can be mapped in S. pombe by Solexa sequencing

Ex vivo expansion of murine marrow cells with interleukin-3 (IL-3), IL-6, IL-11, and stem cell factor leads to impaired engraftment in irradiated hosts

In vitro incubation of bone marrow cells with cytokines has been used as an approach to expand stem cells and to facilitate retroviral integration. Expansion of hematopoietic progenitor cells has been monitored by different in vitro assays and in a few instances by in vivo marrow renewal in myeloablated hosts. This is the first report of studies, using two competitive transplant models, in which cytokine-treated cells, obtained from nonpretreated donors (eg, 5-fluorouracil), were competed with normal cells. A basic assumption is that the expansion of progenitors assayed in vitro as high- and low-proliferative potential colony-forming cells (HPP- and LPP-CFCs) indicates an expansion of stem cells which will repopulate in vivo. This study shows that culture of marrow cells with four cytokines (stem cell factor, interleukin-3 [IL-3], IL-6, IL-11) induces significant expansion and proliferation of HPP-CFC and LPP-CFC. Cell-cycle analysis showed that these hematopoietic progenitors were induced to actively cell cycle by culture with these cytokines. In the first competitive transplant model, which uses Ly5.2/Ly5.1 congenic mice, cytokine-cultured Ly5.2 cells competed with noncultured Ly5.1 cells led to 5% +/- 1% engraftment at 12 weeks and to 4% +/- 2% engraftment at 22 weeks posttransplantation for the cytokine exposed cells. Noncultured Ly5.2 cells competed with cultured Ly5.1 cells led to 70% +/- 1% engraftment at 12 weeks and to 93% +/- 2% engraftment at 22 weeks posttransplantation. In the second model, which uses BALB/c marrow of opposite genders, cultured male cells lead to 13% +/- 9% engraftment at 10 weeks and 2% +/- 1% engraftment at 14 weeks posttransplantation; noncultured male cells lead to 70% +/- 2% and 95% +/- 2% engraftment at 10 and 14 weeks posttransplantation, respectively. Data presented here from two different competitive transplant studies show a defect of cytokine expanded marrow related to cell cycle activation which manifests as defective long-term repopulating capability in irradiated host mice. The engraftment defect is more profound at longer time intervals, suggesting that the most striking effect may be on long-term repopulating cells

TRF3, a TATA-box-binding protein-related factor, is vertebrate-specific and widely expressed

TATA-box-binding protein (TBP) is a highly conserved RNA polymerase II general transcription factor that binds to the core promoter and initiates assembly of the preinitiation complex. Two proteins with high homology to TBP have been found: TBP-related factor 1 (TRF1), described only in Drosophila melanogaster, and TRF2, which is broadly distributed in metazoans. Here, we report the identification and characterization of an additional TBP-related factor, TRF3. TRF3 is virtually identical to TBP in the C-terminal core domain, including all residues involved in DNA binding and interaction with other general transcription factors. Like other TBP family members, the N-terminal region of TRF3 is divergent. The TRF3 gene is present and expressed in vertebrates, from fish through humans, but absent from the genomes of the urochordate Ciona intestinalis and the lower eukaryotes D. melanogaster and Caenorhabditis elegans. TRF3 is a nuclear protein that is present in all human and mouse tissues and cell lines examined. Despite the highly homologous TBP-like C-terminal core domain, gel filtration analysis indicates that the native molecular weight of TRF3 is substantially less than that of TFIID. Interestingly, after mitosis, reimport of TRF3 into the nucleus occurs subsequent to TBP and other basal transcription factors. In summary, TRF3 is a highly conserved vertebrate-specific TRF whose phylogenetic conservation, expression pattern, and other properties are distinct from those of TBP and all other TRFs

Endogenous siRNAs derived from transposons and mRNAs in Drosophila somatic cells

Small interfering RNAs (siRNAs) direct RNA interference (RNAi) in eukaryotes. In flies, somatic cells produce siRNAs from exogenous double-stranded RNA (dsRNA) as a defense against viral infection. We identified endogenous siRNAs (endo-siRNAs), 21 nucleotides in length, that correspond to transposons and heterochromatic sequences in the somatic cells of Drosophila melanogaster. We also detected endo-siRNAs complementary to messenger RNAs (mRNAs); these siRNAs disproportionately mapped to the complementary regions of overlapping mRNAs predicted to form double-stranded RNA in vivo. Normal accumulation of somatic endo-siRNAs requires the siRNA-generating ribonuclease Dicer-2 and the RNAi effector protein Argonaute2 (Ago2). We propose that endo-siRNAs generated by the fly RNAi pathway silence selfish genetic elements in the soma, much as Piwi-interacting RNAs do in the germ line

A genome-wide shRNA–based synthetic interaction screen identifies candidate genes preferentially required for proliferation of p53− cells.

<p>(A) Schematic summary of the screen. p53+ and p53− HCT116 cells were infected in parallel with a pooled lentiviral human shRNA library. The shRNA population was analyzed by massively parallel sequencing at 40 hours and 10 days post-infection (p.i.). (B) Volcano plot. The horizontal and vertical lines indicate the selection criteria. The red points represent shRNAs diminished ≥4-fold in p53− HCT116 cells and ≤2-fold in p53+ HCT116 cells at 10 days p.i. relative to 40 hours p.i. Blue points represent shRNAs diminished in both p53+ and p53− cells, and black points represent shRNAs not diminished in either p53+ or p53− cells. (C) Colony formation assay. p53+ and p53− HCT116 cells infected with a lentivirus expressing individual candidate shRNAs were selected with puromycin and plated in a serial dilution series in 6-well plates. Only one dilution set is shown. Colonies were fixed and stained with crystal violet. Control refers to the empty lentiviral vector, pGIPZ. (D) Proliferation assay. p53+ and p53− HCT116 cells infected with a lentivirus expressing each individual candidate shRNA, or as a control a non-silencing (NS) shRNA, were selected with puromycin and cell proliferation determined by an Alamar Blue fluorescence assay. The results were normalized to that obtained with a NS shRNA, which was set to 1. Error bars represent SD. (E) Proliferation of p53+ and p53− HCT116 cells transfected with an siRNA directed against an individual candidate gene, or a control lamin A/C (LMNA) siRNA, was determined by an Alamar Blue fluorescence assay. The results were normalized to that obtained with the control shRNA, which was set to 1. Error bars represent SD.</p

ETV1 and ATR are preferentially required for proliferation of diverse p53− cell lines.

<p>(A) Proliferation of p53+ and p53− RKO cells expressing an individual candidate shRNA, or as a control a NS shRNA, was determined by an Alamar Blue fluorescence assay. The results were normalized to that obtained with the NS shRNA, which was set to 1. Error bars represent SD. ShRNAs that preferentially affect the proliferation of p53− relative to p53+ cell lines are indicated in red. (B) Proliferation of p53+ and p53− A549 cells transfected with a candidate siRNA, or as a control a LMNA siRNA, was determined by an Alamar Blue fluorescence assay. The results were normalized to that obtained with the LMNA siRNA, which was set to 1. Error bars represent SD. ShRNAs that preferentially affect the proliferation of p53− relative to p53+ cell lines are indicated in red. (C) Proliferation of A549, NCI-H460, NCI-H1299 and NCI-H522 cells expressing a candidate shRNA, or as a control an NS shRNA, was determined by an Alamar Blue fluorescence assay. The results were normalized to that obtained with the NS shRNA, which was set to 1. Error bars represent SD. ShRNAs that preferentially affect the proliferation of p53− relative to p53+ cell lines are indicated in red. (D) p53+ and p53− HCT116 cells expressing an ATR or ETV1 shRNAs or as a control a NS shRNA, were subcutaneously injected into opposite flanks of the same nude mouse, and tumor volume was measured after 4 weeks. The results were normalized to that obtained in p53+ cells expressing a NS shRNA, which was set to 1. Error bars represent SD. * <i>P</i>≤0.0001.</p